The Rank ligand (RANKL)/OPG/RANK biochemical axis has been successfully targeted to treat osteoporosis, rheumatoid arthritis, cancer-induced bone destruction, metastasis, hypercalcemia, and pain (Hofbauer et al., Cancer 92(3): p. 460-470 (2001). Therapies utilizing OPG (Honore et al., Nature Medicine 6(5):521-528 (2000)), an antibody directed to RANKL, or the soluble RANK-Fc protein (Oyajobi et al., Cancer Res 61(6): p. 2572-8 (2001)) are also in development. OPG and soluble RANK-Fc protein constructs bind to RANKL, thereby decreasing amount of RANKL that is available for RANK receptor activation.
In addition to being important in bone biology, RANKL plays a role in the immune system by regulating antigen-specific T cell responses (Anderson et al., Nature 390(6656):175-9 (1997)). RANKL is highly expressed on activated T cells while the RANK receptor is expressed at high levels on mature dendritic cells (DC). The interaction between RANKL and RANK acts as a costimulatory signal, which enhances DC survival and T cell proliferation by inducing DC differentiation, cytokine production and reduced apoptosis in both cell types. Immunotherapy to produce tolerance to transplanted tissues and/or organs can be achieved by blocking the costimulatory signal using RANK antagonists. Blocking costimulation prevents T cell activation by DCs, and causes alloreactive T cells to become anergic and/or undergo apoptosis (Adler et al., Current Opinion in Immunology 14:660-665 (2002)). By a similar mechanism of action, antagonizing RANK signaling could be a treatment for autoimmune and immune-mediated inflammatory disorders such as systemic lupus erythematosus, inflammatory bowel disease, diabetes, multiple sclerosis, rheumatoid arthritis, and ankylosing spondylitis.
Osteoprotegerin (OPG) is a protein of the Tumour Necrosis Factor (TNF) receptor family, and was first described by Simonet et al. (Cell, 89, 309-319 (1997)).
OPG appears to be a crucial element in regulating the natural processes of bone production and turnover. Changes in the balance between OPG and its target receptor RANKL have been noted in a number of conditions associated with abnormal bone metabolism.
OPG has undergone preclinical and clinical testing with potential for application in various conditions associated with increased bone turnover and bone loss, including osteoporosis, rheumatoid arthritis, Paget's disease, periodontal disease, vascular disease and cancers that are located in or have metastasised to bone (For review, see Lorenz et al., J. Amer Med Assoc 292: 490-5 (2004) and references therein). Extensive animal testing of OPG is reported in the literature, indicating equal or superior performance compared with other therapies currently used for reducing bone loss (Simonet et al., Cell, 89, 309-319 (1997); Bolon et al., Cell Mol Life Sci 59, 1569-76 (2002)). OPG has also been shown to reduce pain associated with bone cancers (Luger et al., Cancer Research 61, 4038-4047 (2001)).
OPG is described in detail in U.S. Pat. No. 6,015,938, U.S. Pat. No. 6,284,740, U.S. Pat. No. 6,284,728, U.S. Pat. No. 6,613,544, U.S. Pat. No. 6,316,408, U.S. Pat. No. 6,288,032 and U.S. Pat. No. 6,369,027. Transgenic mice lacking expression of OPG are described in U.S. Pat. No. 6,087,555.
OPG has undergone Phase I trials in both multiple myeloma patients and in osteoporosis patients for reduction of bone turnover. In preliminary Phase I/II studies in post-menopausal women suffering from osteoporosis, a single injection of OPG was found to suppress markers of bone turnover by 30-80% for several days (Bekker et al., J. Bone Miner Res 16, 348-360 (2001)). Multiple myeloma patients were treated with a range of doses of OPG, and showed up to 50% decrease in bone resorption over 30 or more days (Body et al., Cancer 97, 887-892 (2003)). Thus there is very strong evidence, both from preclinical studies and clinical studies, that OPG is likely to be a highly effective treatment in a variety of conditions characterised by abnormal bone metabolism.
The tumour necrosis factor (TNF) family of cytokines and their corresponding receptors contains a considerable number of members and there is substantial cross reactivity among members in ligand-receptor interactions (for review, see Igney, F. H. and Krammer, P. H Nature Reviews Cancer 2, 277-288 (2002)). TNF receptors generally trigger one of two kinds of response, either an apoptotic (programmed cell death) response or an effect on cell metabolic pathways via activation of the transcription factor NFkB. The receptor family also contains members which act as decoys, to reduce the effectiveness of interaction between other family ligand-receptor pairs. OPG appears to fall into this receptor class.
Compared with many TNF receptor family members, OPG is relatively restricted in its target specificity. According to current knowledge, OPG binds to only two TNF-like ligands:                1. RANKL (Receptor activator of NFkB ligand), a TNF-like cell surface molecule normally interacts with a TNF receptor family member, RANK, which is expressed on osteoclast precursors, dendritic cells T-cells and haematopoietic precursors (Kong et al., Nature 397:315-323 (1999). RANKL interacts with RANK on cell surfaces to stimulate the production and activity of osteoclasts, the principal cells involved in bone turnover (Hsu, H et al., Proc Natl Acad Sci USA 96, 3540-3545 (1999)). RANKL is expressed on stromal cells/osteoblasts (Kong et al., Nature 397:315-323 (1999)). The interaction of OPG with RANKL inhibits RANKL's ability to attach to RANK and stimulate osteoclasts and it is this activity of OPG that confers its ability to reduce bone loss (Lacey et al., Amer J Pathol 157, 435-448 (2000); Simonet et al., Cell, 89, 309-319 (1997)). The binding constant of dimeric OPG binding to RANKL has been reported as 6.7 nM (Willard et al., Protein Expr. Purif 20, 48-57 (2000)) with other reports placing the binding constant between 2 and 10 nM.        2. TRAIL (TNF-related apoptosis inducing ligand), a TNF-like cell surface molecule involved in the induction of apoptosis in cancer cells. OPG is seen as one of a small number of decoy receptors for TRAIL, acting to modulate its ability to target cancer cells (Reviewed in Igney and Krammer (2002) as referenced above). OPG would thus be expected to enhance cancer cell survival if present at a relevant site in sufficient quantities, and its ability to increase the survival of tumour cells has been documented (for example, Neville-Webbe et al., Breast Cancer Res Treat. 86, 271-82 (2004); Holen I et al., Cancer Res 62, 1619-1623 (2002)). The affinity of OPG for TRAIL has been reported as 3 nM (Emery et al., J. Biol. Chem. 273, 14363-7 (1998)) although this may vary with temperature (Truneh et al., J. Biol. Chem. 275, 23319-25 (2000)).        
The ability of OPG to inhibit TRAIL activity, resulting in a possible increase in risk of cancer development, has been noted in the medical literature as a likely deterrent to the use of this agent as a therapeutic. Among the application areas for agents inhibiting bone loss are conditions such as osteoporosis and Paget's disease, which are not in themselves life threatening, but require long term therapy. Furthermore, one of the most important potential uses of OPG is in adjunct treatment of cancer patients with bone metastases. In all of these potential application areas, any indication of increased growth and survival of cancer cells associated with treatment would be a deterrent to use. Hence there is a need to generate a novel variant of OPG that substantially lacks TRAIL-binding capabilities. Such a variant would lack ability to significantly interfere with natural anti-cancer mechanisms while retaining its ability to interfere with the RANK/RANKL interaction that stimulates osteoclast formation and activity, and consequent bone loss.